Antimicrobial guanidinium macromolecules with bacteria targeting moieties

ABSTRACT

Compositions and methods regarding antimicrobial guanidinium macromolecules with one or more targeting moieties for selectively targeting bacteria are provided. According to an embodiment, an antimicrobial macromolecule is provided that comprises a polymer backbone and one or more guanidinium moieties that extend from the polymer backbone. The antimicrobial macromolecule further comprises a targeting moiety that extends from the polymer backbone. The targeting moiety can comprise a substance favored for consumption by bacteria, such as a monosaccharide.

TECHNICAL FIELD

The subject disclosure relates to antimicrobial macromolecules and moreparticularly to one more guanidinium macromolecules with one or moretargeting moieties for selectively targeting bacteria.

SUMMARY

The following presents a summary to provide a basic understanding of oneor more embodiments of the invention. This summary is not intended toidentify key or critical elements, or delineate any scope of theparticular embodiments or any scope of the claims. Its sole purpose isto present concepts in a simplified form as a prelude to the moredetailed description that is presented later. In one or more embodimentsdescribed herein, methods and/or compositions regarding antimicrobialguanidinium macromolecules with one or more targeting moieties forselectively targeting bacteria are described.

According to an embodiment, an antimicrobial macromolecule is provided.The antimicrobial macromolecule can comprise a polymer backbone, one ormore guanidinium moieties that extend from the polymer backbone, and atargeting moiety that extends from the polymer backbone. The targetingmoiety can comprise a substance favored for consumption by bacteria,such as a monosaccharide. For example, in one or more implementations,the targeting moiety is selected from a group consisting of mannose andglucose. In some implementations, the polymer backbone of theantimicrobial macromolecule comprises polycarbonate. In otherimplementations, the polymer backbone can be selected from a groupconsisting of polylysine, polyionene, and polyethylene imine.

The subject antimicrobial macromolecule is highly effective at killingGram-negative bacteria and Gram-positive bacteria. For example, invarious example implementations, the antimicrobial macromolecule iseffective at killing multi-drug resistant (MDR) bacteria selected from agroup consisting of: Acinetobacter baumannii, Klebsiella pneumonia,Escherichia coli, Staphylococcus aureus, methicillin-resistantStaphylococcus aureus, and Pseudomonas aeruginosa. The antimicrobialmacromolecule further exhibits low toxicity in mammalian cellsdemonstrated by a red blood cell viability level greater than 95% at aneffective dose. Moreover, the antimicrobial macromolecule is attributedto less than a 1% development of bacterial antimicrobial agentresistance at an effective dose over at least 20 passages.

In another embodiment a method is provided. The method can comprisedissolving a monosaccharide with a guanidinium functionalized monomer ina solvent, and polymerizing the guanidinium functionalized monomer toform a polymer, wherein the polymer comprises a plurality of covalentlybonded units of the guanidinium functionalized monomer and a least twoperipheral ends, wherein at least one unit of the monosaccharide iscovalently bound to at least one of the peripheral ends. In one or moreimplementations, the monosaccharide comprises a protected monosaccharideand wherein the guanidinium functionalized monomer comprises cycliccarbonate with a protected guanidinium moiety bound to the cycliccarbonate via a spacer group. In accordance with these implementations,the polymerizing comprises an organocatalyzed ring openingpolymerization of the cyclic carbonate using the protectedmonosaccharide as an initiator. The method can further comprise removingprotection groups from the protected monosaccharide and the protectedguanidinium moiety, thereby forming a deprotected polymer, wherein thedeprotected polymer is effective at killing Gram-negative bacteria andGram-positive bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a diagram of an example, non-limiting chemicalstructure that can characterize one or more antimicrobial guanidiniummacromolecules in accordance with one or more embodiments describedherein.

FIG. 2 illustrates chemical formulas of example, non-limiting protectedmonosaccharides that can be incorporated with one or more antimicrobialguanidinium macromolecules in accordance with one or more embodimentsdescribed herein.

FIG. 3 illustrates chemical formulas of example, non-limitingguanidinium moieties comprising side-chains that can be incorporated asspacer groups in one or more antimicrobial guanidinium macromolecules inaccordance with one or more embodiments described herein.

FIG. 4A illustrates a diagram of an example, non-limiting synthesisscheme that can facilitate generation of one or more guanidiniummonomers in accordance with one or more embodiments described herein.

FIG. 4B illustrates a diagram of an example, non-limiting synthesisscheme that can facilitate generation of one or more guanidiniumpolymers in accordance with one or more embodiments described herein.

FIG. 5 presents an example, non-limiting table providing the cumulativedistribution of minimum inhibitory concentration (MIC) values of variousantimicrobials, including the subject antimicrobial guanidiniummacromolecules, against clinically isolated. MDR (MDR) Acinetobacterbaumannii (A. baumannii) in accordance with one or more embodimentsdescribed herein.

FIG. 6 presents an example, non-limiting table comparing the MIC valuesof various antimicrobials, including the subject antimicrobialguanidinium macromolecules, against different strains of clinicallyisolated bacterial in accordance with one or more embodiments describedherein.

FIG. 7 illustrates an example, non-limiting graph that depicts hemolysisactivity of one or more antimicrobial guanidinium macromolecules inaccordance with one or more embodiments described herein.

FIG. 8 illustrates an example, non-limiting graph that demonstrates therobustness of one or more antimicrobial guanidinium macromoleculesagainst inducing antimicrobial agent resistance in accordance with oneor more embodiments described herein.

FIG. 9 presents an example, non-limiting table demonstrating theeffective dose evaluation of various antimicrobials, including thesubject antimicrobial guanidinium macromolecules, against A. baumanniiin a blood infected mouse model, accordance with one or more embodimentsdescribed herein.

FIG. 10 presents an example, non-limiting graph demonstrating theefficacy of various antimicrobials, including the subject antimicrobialguanidinium macromolecules, against A. baumannii in a blood infectedmouse model, accordance with one or more embodiments described herein.

FIG. 11 illustrates another example, non-limiting graph thatdemonstrates the efficacy of various antimicrobials, including thesubject antimicrobial guanidinium macromolecules, against A. baumanniiin a blood infected mouse model, accordance with one or more embodimentsdescribed herein.

FIG. 12 illustrates a flow diagram of an example, non-limiting methodthat can facilitate generating one or more antimicrobial guanidiniummacromolecules in accordance with one or more embodiments describedherein.

FIG. 13 illustrates a flow diagram of another example, non-limitingmethod that can facilitate generating one or more guanidiniummacromolecules in accordance with one or more embodiments describedherein.

FIG. 14 illustrates a flow diagram of an example, non-limiting methodthat can facilitate killing of a pathogen with one or more guanidiniummacromolecules in accordance with one or more embodiments describedherein.

DETAILED DESCRIPTION

The following detailed description is merely illustrative and is notintended to limit embodiments and/or application or uses of embodiments.Furthermore, there is no intention to be bound by any expressed orimplied information presented in the preceding Summary and BriefDescription of the Drawings sections, or in the Detailed Descriptionsection.

Over the last few decades, the emergence ofantibiotic/pharmaceutical-resistance amongst pathogens such asAcinetobacter baumannii (A. baumannii) and Klebsiella pneumonia (K.pneumonia) has become increasingly prevalent. Despite best efforts, thepanacea for these recalcitrant infections has not been found. With noeffective and safe treatments available, MDR infections are fastmorphing into a global healthcare threat. Polymixins remain the lastline treatment for these MDR infections although they are associatedwith significant adverse effects (nephrotoxicity and neurotoxicity) andare ineffective against Gram-positive bacteria. There is thus a direneed to develop novel antimicrobial compounds that have both potent anda broad spectrum of activity (against Gram-positive and Gram-negativebacteria), yet at the same time, well-tolerated with low propensity forresistance development.

The disclosed subject matter is directed the development and applicationof one or more novel guanidinium based antimicrobial macromoleculescapable of selectively targeting and eradicating MDR bacteria, in vitroand in vivo, while minimizing toxicity and resistance onset. In someimplementations, the disclosed guanidinium macromolecules can also beeffective at killing fungi, yeast, and other pathogens. In one or moreembodiments, the subject antimicrobial guanidinium-based macromoleculescan comprise one or more targeting moieties attached to a guanidiniumfunctionalized polymer. The guanidinium functionalized polymer cancomprise a polymer backbone with one or more antimicrobial guanidiniummoieties that extend therefrom. In some implementations, the polymerbackbone can comprise a polycarbonate. However, other suitable polymerscan be employed for the polymer backbone, such as but not limited to,polylysine, polyionene, polyethylene imine and the like. In variousembodiments, the guanidinium functionalized polymer can facilitatekilling bacterial cells via an electrostatic interaction mechanism. Inthis regard, the guanidinium moieties can be cationic (positivelycharged) in nature and configured to bind with and neutralize theanionic (negatively charged) bacterial surface, resulting in a chargeneutralization that allows the macromolecule to translocate through thebacterial membrane (e.g., as a non-polar species). The macromolecule isthen released through the membrane leading to cytosol precipitation andsubsequent bacterial cell apoptosis.

The one or more targeting moieties of the subject guanidiniumfunctionalized polymers can comprise a substance that is favored forconsumption by Gram-negative and Gram-positive bacteria. In this regard,the targeting moiety can selectively attract bacterial cells andfacilitate increasing the efficiency of the attachment of themacromolecule to the bacterial surface and subsequent translocation ofthe guanidinium functionalized polymer through the bacterial cellmembrane. For example, in various embodiments, the one or more targetingmoieties can comprise carbohydrate moieties (e.g., sugars). The sugartargeting moiety has been shown to enhance bacterial targeting andantimicrobial activity of the entire macromolecule, increase solubilityof the macromolecule, and further mitigate toxicity. Moreover, targetingmoieties that comprise sugars have been shown to target the bacteriacell wall/membrane in such a way that the sugar gets taken up andchemically incorporated into the bacterial cell wall/membrane.Accordingly, in addition to facilitating targeting selectivity andantimicrobial efficiency of the guanidinium based macromolecule, thesugar moiety can further contribute to local disorder and stress withinthe lipid bilayer of the bacterial cell, leading to the membrane damageand thus a higher rate of cell lysis/apoptosis. This approach may alsobe used to engineer the wall/membrane for smart and targeted therapiesor diagnostics.

Some example compounds comprising a polycarbonate polymer backbonefunctionalized with guanidinium moieties and further comprising a singleunit of glucose or mannose as the targeting moiety were tested in vilroand in vivo (in a blood infection animal model) with substantiallygreater success over traditional antibiotics. Relative to leadingantibiotics including ceftriaxone, gentamicin, imipenem, andlevofloxacin, the example compounds exhibited higher efficacy at killingboth Gram-negative and Gram-positive MDR bacteria at substantially lowerminimum inhibitory concentration (MIC) values and lower effective doses.The example compounds also exhibited extremely low toxicity against redblood cells with red blood cell (RBC) hemolysis viability levels at ornear 100%, even at concentrations up to 1000 parts per million (ppm). Inaddition, unlike the commercial antibiotics, the example compoundsfailed to engender antimicrobial resistance over many passages. Thesample compounds also demonstrated increased efficacy, reduced effectivedose amounts, reduced toxicity and strong immunity to development ofantimicrobial resistance relative to guanidinium functionalized polymerswithout the disclosed targeting moieties and associated chemicalstructure.

Additionally, the subject guanidinium-based macromolecules can bebiocompatible, biodegradable, non-hemolytic, and non-cytotoxic atconcentrations above the MIC. The subject guanidinium-basedmacromolecules are therefore attractive for a wide range of consumerproducts, such as for example, antibiotic pharmaceuticals, cosmetics,skin lotions, and the like. The term biodegradable is defined by theAmerican Society for Testing and Materials (ASTM) as degradation causedby biological activity, especially by enzymatic action, leading to asignificant change in the chemical structure of the material. Forpurposes herein, the subject guanidinium-based macromolecules having(and variations thereof) can be characterized as biodegradable becausethey have been shown to undergo at least 60% biodegradation within 180days, in accordance with ASTM D6400. The subject guanidinium-basedmacromolecules can also be characterized as enzymatically biodegradablebecause they have been shown to be degraded (e.g., depolymerized) by areaction catalyzed by an enzyme. A biocompatible material is definedherein as a material capable of performing with an appropriate hosresponse in a specific application.

One or more embodiments are now described with reference to thedrawings, wherein like referenced numerals are used to refer to likeelements throughout. In the following description, for purposes ofexplanation, numerous specific details are set forth in order to providea more thorough understanding of the one or more embodiments. It isevident, however, in various cases, that the one or more embodiments canbe practiced without these specific details.

FIG. 1 illustrates a diagram of an example, non-limiting chemicalStructure 100 that can characterize one or more antimicrobialguanidinium macromolecules in accordance with one or more embodimentsdescribed herein. Structure 100 comprises a polymer backbone with atargeting moiety “R” extending therefrom. In this regard, the targetingmoiety R can be covalently bonded to one or more atoms of the polymerbackbone. In the embodiment shown, the polymer backbone can comprise oneor more polymer units (represented by the series of connected circleswithin parenthesis) respectively including a guanidine-based functionalgroup extending therefrom and covalently bonded to one or more atoms ofthe polymer unit. In this regard, the polymer backbone can comprise oneor more repeat monomer units that are respectively functionalized with aguanidine-based antimicrobial moiety. These monomers are referred toherein as guanidinium functionalized monomers. The guanidiniumfunctional group comprises a guanidine-based antimicrobial moiety thatcan be connected to the polymer backbone via at least one spacer group“S.” The number “n” of repeating and connected/bonded guanidiniumfunctionalized monomer units that form the polymer backbone can vary.For example, in some implementations, the number n of repeating monomerunits can be one or more and one thousand or less. However, in one ormore exemplary embodiments, the number n of repeating monomer units isbetween 5 and 40 units. In another embodiment, the number n of repeatingmonomer units is between 10 and 30 units. Still in yet anotherembodiment, the number n of repeating units is between 15 and 25 units,with an optimal number of units being 20 units.

In the embodiment shown, each monomer unit of the polymer backboneincludes at least one guanidinium functional group that consists of theguanidine-based antimicrobial moiety connected to thereto via the spacergroup S. However, in some implementations, one or more of the monomerunits can include no guanidinium functional groups and/or one or more ofthe monomer units can include two or more guanidinium based functionalgroups. In this regard, no restriction is placed on the polymer skeletalstructure of the skeletal backbone. Exemplary non-limiting polymerskeletal structures can include linear polymers, branched polymers, starpolymers, mykto-arm star polymers, latter polymers, cyclic polymers, andgraft polymers. The forgoing polymer types can comprise a homopolymer, arandom copolymer, or a block copolymer chain. In various exemplaryembodiments, the antimicrobial guanidinium based macromolecule is alinear polymer comprising a plurality of covalently bonded guanidiniumfunctionalized monomer units. Herein, a linear polymer has one branchhaving two peripheral ends (i.e., dangling ends, as the two ends of asegment of a rope). At least one of the peripheral ends can comprise atargeting moiety R covalently bonded thereto. The one branch cancomprise one or more polymer chain segments covalently linked togetherat respective polymer chain ends by way of any suitable linking group,which can include a single bond. Each polymer chain segment of a linearpolymer can comprise a homopolymer, random copolymer, or block copolymerchain comprising one or more repeat units. At least one of the polymerchain segments comprises one or more repeat units of the guanidiniumfunctionalized monomer.

The targeting moiety R, the polymer backbone, the spacer group S and thechemical structure of the guanidine-based antimicrobial moiety can vary.In various embodiments, the targeting moiety R can include a substancethat is favored for consumption by bacteria. In this regard, thetargeting moiety R can selectively attract bacteria to facilitate theconsumption and translocation of the subject guanidinium-basedmacromolecules into the bacterial cytosol and/or integration of thetargeting moiety R into the bacterial cell wall/membrane. In one or moreembodiments, the targeting moiety R can comprise one or morecarbohydrates (also referred to as saccharides). For example, in someimplementations, the targeting moiety R can comprise one or more sugars,including monosaccharides and disaccharides. Some suitable sugars forthe targeting moiety R can include but are not limited to, fructose,galactose, glucose, and/or mannose. In some implementations, thetargeting moiety R can be a single unit of sugar. As exemplified infra,some example guanidinium-based macromolecules having Structure 100 andemploying only a single unit of sugar for the targeting moiety R havedemonstrated substantially higher antimicrobial efficacy relative toguanidinium functionalized polymers without a targeting moiety R, aswell leading antibiotics including ceftriaxone, gentamicin, andimipenem. In other implementations, the targeting moiety R can includeplurality of sugar units.

In some embodiments, the targeting moiety R can comprise or be initiatedfrom a protected monosaccharide. Protected monosaccharides includesugars in which at least one hydroxyl group is protected by some form ofmodification. With these embodiments, usage of a protected sugar as thetargeting moiety R (or to initialize the targeting moiety R) canminimize the synthesis complexity of the subject macromolecules inimplementations in which the monomer employed for the polymer backbonecomprises a cyclic carbonate bearing one or more protected guanidiniumfunctional groups.

FIG. 2 illustrates chemical formulas of some example, non-limitingprotected monosaccharides that can be incorporated with one or moreantimicrobial guanidinium macromolecules as the targeting moiety R, orto initialize the targeting moiety R, in accordance with one or moreembodiments described herein. The example protected monosaccharides 201,202, 203 and 204 shown in FIG. 2 respectively include2,3:5,6-Di-O-isopropylidene-a-D-mannofuranose,1,2:3,4-Di-O-isopropylidene-alpha-D-galactopyranose,2,3:4,5-Di-O-isopropylidene-beta-D-fructopyranose, and1,2:5,6-Di-O-isopropylidene-alpha-D-glucofuranose. It should beappreciated that these protected sugars are merely exemplary and thatother protected sugars can be employed as the targeting moiety R of thesubject antimicrobial guanidinium based macromolecules.

With reference back to FIG. 1, in one or more embodiments, the polymerbackbone can comprise polycarbonate. In other embodiments, the polymerbackbone can comprise polylysine, polyionene, polyethylene imine and thelike.

In one or more exemplary embodiments, the polymer backbone can be formedusing a ring-opening polymerization of a cyclic carbonate monomerbearing one or more protected guanidinium functional groups. Forexample, in some implementations, the cyclic carbonate monomer caninclude methyltetrachloride (MTC) hydroxide (OH). (MTC-OH). With theseembodiments, the subject antimicrobial guanidinium macromolecules canhave a chemical structure characterized by Structure 100 shown in FIG.1, and further characterized by Structure I below:

In Structure I, the guanidine-based antimicrobial moiety is connected tothe polymer backbone via an ethyl spacer group S. However, the spacergroup S of Structure 100 and the related structures described herein(e.g., Structure, Structure II, and the like), can vary. For example, inone or more additional embodiments, the spacer group S can include butis not limited to: a propyl group, a butyl group, a pentyl group, acyclohexyl group, a phenyl group, or a benzyl group. In anotherembodiment, the spacer group S can include an isopropyl group. With thisembodiment, the two guanidine-based antimicrobial moieties can be boundto the isopropyl group, supporting a Di-guanidinium functional groupthat extends from the polymer backbone.

FIG. 3 illustrates chemical formulas of some example, non-limitingguanidinium moieties comprising side-chains that can be incorporated asthe spacer group S of one or more antimicrobial guanidiniummacromolecules in accordance with one or more embodiments describedherein. The example guanidinium moieties are shown with various sidechains bound to one or more guanidine compounds via single covalentbound to a nitrogen atom of the guanidinium moiety. For example, Formula301 depicts a guanidinium moiety with either an ethyl, propyl, butyl, orpentyl side chain (e.g., as indicated by the number of carbons “n” being2-5). Formula 302 depicts a Di-guanidinium moiety with an isopropyl sidechain. Formula 303 depicts a guanidinium moiety with a phenyl sidechain, and Formula 304 depicts a guanidinium moiety with a benzyl sidechain. The oxygen atoms shown with dashed lines in Formulas 301, 302,303 and 304 respectively represent the portion of the polymer backboneof the subject guanidinium-based macromolecules to which the side groupscan connect (e.g., vi a single bond). It should be appreciated howeverthat the manner in which the example side chains are bound to thepolymer backbone and the atom or atoms to which the side chains arebound in the subject antimicrobial guanidinium-based macromoleculeshaving Structure 100 (and variations thereof) can vary.

With reference to FIGS. 1-3, in view of various optional spacer groupvariations that can be employed for the spacer group S of Structure 100,in some embodiments, one or more antimicrobial guanidiniummacromolecules described herein can be characterized by Structure 100and further characterized by Structure II below:

wherein X is a spacer group selected from a group consisting of: apropyl group, a butyl group, a pentyl group, a cyclohexyl group, aphenyl group, and a benzyl group. For example, in some embodiments, oneor more antimicrobial guanidinium macromolecules described herein can becharacterized by Structure 100 and further characterized by StructuresIII or IV below:

In Structures 100, I, II, III and IV, the quinine-based antimicrobialmoiety is shown as a neutral structure, as represented by Formula Ibelow.

However, in some embodiments, the quinine-based antimicrobial moiety canbe cationic in nature so as facilitate the charge neutralization withanionic surfaces of bacterial membranes, thereby facilitatingtranslocation of the resulting nonpolar macromolecule through thebacterial membrane. In this regard, in some embodiments, theantimicrobial moiety can comprise a hydrosalt of a guanidiniumfunctional group with a positively-charged protonated form of theguanidinium group that is ionically associated with a negatively-chargedcounterion. For example, in one or more implementations, such ahydrosalt of a quinidine group can be represented by Formula II below:

wherein X′ is a negative-charged counterion. For example, in one or moreimplementations, the negative-charged counterion X′ can comprisetrifluoroacetic acid (TFA). With these implementations, thequinine-based antimicrobial moiety of the subject guanidinium basedmacromolecules can be a cationic guanidinium moiety represented byFormula III or Formula IV as follows:

wherein TFA⁻ represents trifluoroacetic acid, the negatively chargedcounterion.

In various embodiments in which a neutral guanidinium moiety havingchemical Formula I is described or depicted throughout the subjectdisclosure (e.g., in accordance with Structure 100 of FIG. 1 andStructures I, II III and IV above), the neutral guanidinium moiety canbe interchanged for a cationic guanidinium moiety such as thoserepresented by Formulas II, III, IV and the like, (and vice versa).

FIG. 4A illustrates a diagram of an example, non-limiting synthesisscheme 400 that can facilitate generation of one or more guanidiniumfunctionalized monomers in accordance with one or more embodimentsdescribed herein. The one or more guanidinium functionalized monomersgenerated via synthesis scheme 400 can be further polymerized togenerate one or more of antimicrobial guanidinium macromolecules inaccordance with one or more embodiments described herein. For example,synthesis scheme 400 can be employed to produce one or more guanidiniumfunctionalized monomers that can be used to generate one or moreantimicrobial macromolecules characterized by Structure 100, Structure Iand the like. Repetitive description of like elements employed in otherembodiments described herein is omitted for sake of brevity.

In the embodiment shown, the resulting guanidinium functionalizedmonomer having Structure 408 is referred to herein as MTC-C₂GuanBoc. Theguanidinium functionalized monomer MTC-C₂GuanBoc, comprises a cycliccarbonate (MTC) with a protected guanidinium functional moietycovalently bonded thereto via an ethyl spacer group. In this regard, theguanidinium moiety comprises two protecting tert-butyloxycarbonyl (tBocor Boc) groups. The Boc groups can correspond to independent acid-liableprotecting groups. Although the protecting groups of Structure 408 areBoc groups, other suitable protecting groups can be employed, such asbut not limited to, benzyloxycarbonyl (Bnoc), and fluorenyloxycarbonyl(Fmoc).

In accordance with synthesis scheme 400 the MTC-C₂GuanBoc monomer can beprepared using by reacting one or more cyclic carbonates havingStructure 402 with one or more protected guanidinium compounds havingStructure 406 using a suitable amount of reagent oxalyl chloride 404.For example, in one implementation, synthesis scheme 400 was used togenerate the Boc-protected guanidine-functionalized cyclic carbonatemonomer (Boc-GC) having Structure 408 as follows. In a dry three-neckcircular bottom flask (250 milliliters (mL)) equipped with a stir bar,MTC-OH (3.64 grams (g), or 22.75 millimoles (mmol)) was dissolved in drytetrahydrofuran (THF). (50 mL) with 3-4 drops of dimethylformamide(DMF). A solution of oxalyl chloride (2.90 mL, 33.9 mmol) in dry THF (50mL) was added dropwise to the above solution under an inert atmosphere,and the reaction continued for 1.0 hour until the addition of oxalylchloride was completed. Then, a constant stream of nitrogen gas wasbubbled through the reaction mixture for approximately 1.0 h to removeTHF and other volatiles. The residue was dried under high vacuum to givea yellowish solid (i.e. 5-chlorocarboxy-5-methyl-1.3-dioxan-2-oneintermediate). The solid was heated to 60 degrees Celsius (° C.) for 2-3minutes to further remove any residual volatiles, and then re-dissolvedin dry CH₂Cl₂ (50 mL) and cooled down to 0° C. via an ice bath under anitrogen atmosphere. A mixture of Boc-Gua-OH (6.94 g, 21.0 mmol) andpyridine (1.84 mL, 22.9 mmol) dissolved in dry CH₂Cl₂ (50 mL) was thenadded dropwise over a duration of 30 minutes and allowed to stir at 0°C. for an additional 30 minutes before letting it react at ambienttemperature overnight. After removal of the solvent, the crude productwas subjected to purification by flash column chromatography usingsilica gel and a hexane-ethyl acetate solvent system as the eluent(gradient elution up to 50% volume ethyl acetate) to yield Boc-GC as awhite powder (6.1 g, 88% yield). The results of a proton nuclearmagnetic resonance (¹H NMR) study of the Boc-GC at 400 megahertz (MHz),in deuterated chloroform (CDCl₃) at 22 degrees Celsius (° C.), were asfollows: δ 11.47 (s, 1H, —NH—), 8.33 (s, 1H, —NH—), 4.65 (d, 2H,—CH₂OCOO—), 4.20 (m, 4H, —CH₂OCOO— and —OCH₂—), 3.42 (dd, 2H, —CH₂N—),1.62-1.70 (m, 4H, —CH₂—), 1.47 (s, 18H, Boc-CH₃), 1.31 (s, 3H, —CH₃).

In another implementation, synthesis scheme 400 was used to generateanother guanidinium functionalized monomer Boc-protected guanylatedbutanol (Boc-Gua-OH) as follows,1,3-Bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (6.6 g, 23.2mmol) was dissolved in 20 mL of dry CH₂C₁₂, and the solution was addedto the mixture of 4-amino-1-butanol (4.2 mL, 44.6 mmol) andN,N-diisopropylethylamine (12.0 mL, 68.8 mmol). The reaction mixture wasleft to stir overnight at room temperature. Upon reaction completion, aconstant stream of nitrogen gas was bubbled through the reaction mixturefor approximately 1.0 hour to purge the gaseous by-product. MeSH. Afterthe removal of residual solvent in vacuo, the crude product was purifiedby flash column chromatography using silica gel and a hexane-ethylacetate solvent system as the eluent (gradient elution up to 50% vol,ethyl acetate) to yield the Boc-protected guanylated butanol(Boc-Gua-OH) as a white powder (7.0 g, 95% yield). The results of a ¹HNMR (400 MHz, CDCl₃, 22° C.) were as follows: δ 11.48 (s, 1H, —NH—),8.42 (s, 1H, —NH—), 3.70 (dd, 2H, HOCH₂—), 3.46 (dd, 2H, —CH₂NH—), 1.65(m, 4H, —CH₂—), 1.50 (d, 18H, Boc-CH₃).

While one or more particular reactants (e.g., cyclic carbonate havingStructure 402, protected guanidinium compounds having Structure 406,etc.), reagents, and/or solvents are depicted; additional embodiments ofsynthesis scheme 400 are also envisaged. For example, the principalmechanisms of synthesis scheme 400 can be applied to various carbonatebased homopolymers, polylysine based homopolymers, polyionene basedhomopolymers, polyethylene based homopolymers, and various guanidiniumcompounds (e.g., comprising one or more guanidinium moieties havingvarious alternative side chains, such as those described with referenceto FIG. 3 and the like), in accordance with the various featuresdescribed herein.

FIG. 4B illustrates a diagram of an example, non-limiting synthesisscheme 401 that can facilitate generation of one or more antimicrobialguanidinium macromolecules in accordance with one or more embodimentsdescribed herein. In this regard, synthesis scheme 401 can be employedto produce one or more antimicrobial polymers characterized by Structure100. Structure I and the like. Repetitive description of like elementsemployed in other embodiments described herein is omitted for sake ofbrevity.

Synthesis scheme 401 can be employed to install various small moleculemonosaccharides onto a guanidinium functionalized polymer backbone usingvarious protected monosaccharides as the initiator 410. For example, insome embodiments, the protected monosaccharide can be D-mannosediacetonide. Other suitable protected monosaccharides for the initiator410 can include but are not limited to, diacetone-D-galactose,diacetone-D-glucose, and diacetone-D-fructose. Synthesis scheme 401particularly exemplifies the generation of one or more antimicrobialguanidinium macromolecules prepared by organocatalyzed ring openingpolymerization (OROP) of one or more guanidinium functionalized monomersMTC-C₂GuanBoc having Structure 408. In accordance with synthesis scheme401, the OROP of MTC-C₂GuanBoc can be performed using a protectedmonosaccharide as the initiator 410 (and corresponding targetingmoiety), and one or more reagents 412. For example, the reagents 412 caninclude but are not limited to, an organo-catalyst (e.g.1,8-Diazabicyclol[5,4,0]-undec-7-ene (DBU),N-(3,5-trifluoromethyl)phenyl-N-cyclohexylthiourea (TU), or DBU/TU), asolvent (e.g., methylene chloride (CH₂CL₂)), and optionally, anaccelerator.

The OROP of the one or more guanidinium functionalized monomersMTC-C₂GuanBoc produces an intermediate protected homopolymer havingStructure 414. The intermediate protected homopolymer comprises apolymer backbone consisting of a plurality of repeat carbonate unitsrespectively comprising protected guanidinium groups extending therefrom(e.g., wherein the number of repeat units corresponds to the variable“n” of Structure 100 and the like). In various embodiments, the numberof repeat units is between 5 and 65, however it should be appreciatedthat any number of repeat units greater than one and less than onethousand are envisioned. In particular, due to the exquisite level ofcontrol of OROP, synthesis scheme 401 can be employed to synthesizeprotected homopolymer having Structures 414 (and the like) to desiredrepeat unit lengths based on the initial monomer to initiator (e.g.,MTC-C₂GuanBoc to protected monosaccharide) feed ratio. The intermediateprotected homopolymer further comprises a protected sugar groupcovalently bonded to a peripheral end of the polymer backbone.

Subsequent deprotection 416 of the intermediate protected homopolymerforms a resultant antimicrobial polymer having Structure 418. Thedeprotection of the protected homopolymer at 416 can be performed usingtrifluoroacetic acid (TFA), or another suitable protic acid. Theresultant antimicrobial polymer comprises one or more guanidiniumfunctional groups respectively attached to a polymer backbone and amonosaccharide targeting moiety that extends from a peripheral end ofthe polymer backbone. In this regard, the chemical Structure 418 of theresultant antimicrobial guanidinium macromolecule generated viasynthesis scheme 401 corresponds to Structure 100 and Structure I,wherein the antimicrobial moiety R comprises a single monosaccharideunit, and the polymer backbone comprises a plurality of guanidiniumfunctionalized carbonate units respectively attached thereto via anethyl spacer group S.

In various exemplary implementations, synthesis scheme 401 was performedseparately using four different protected sugars, including D-mannosediacetonide, diacetone-D-galactose, diacetone-D-glucose, anddiacetone-D-fructose, to generate four different intermediate protectedhomopolymers (e.g., having Structure 414 or the like) and subsequentprotected antimicrobial macromolecules (e.g., having Structure 418 orthe like) with different sugar targeting moieties R. The protectedpolymers are respectively referred to herein as mannose-C₂GuanBoc,galactose-C₂GuanBoc, glucose-C₂GuanBoc. and fructose-C₂GuanBoc. Therespective protected polymers mannose-C₂GuanBoc, galactose-C₂GuanBoc,glucose-C₂GuanBoc. and fructose-C₂GuanBoc were characterized bycomparing various integrated intensities of the ¹H resonances fromprotons of the sugar initiators relative to the backbone and pendantgroups of the Boc protected guanidine monomer. The protected polymersmannose-C₂GuanBoc, and galactose-C₂GuanBoc, glucose-C₂GuanBoc exhibitednarrow molecular weight distribution with a polydispersity index rangingbetween 1.16 and 1.20 based on gel permeation chromatography prior topost functionalization deprotection 416.

For example, in one or more embodiments, mannose-C₂GuanBoc was preparedin accordance synthesis scheme 401 and the following protocol.Initially, the OROP of MTC-C₂GuanBoc with D-mannose diacetonide wasperformed at a molar ratio of 25:1. In this regard, mannose-C₂GuanBocwas prepared by dissolving D-mannose diacetonide (e.g., at 12 milligrams(mg), or 0.045 millimoles (mmol)) together with TU (e.g., at 20 mg, or0.056 mmol) and MTC-C₂GuanBoc (e.g., at 0.5 grams (g), or 1.12 mmol) in2.0 milliliters (ml) of CH₂Cl₂. Upon complete dissolution. DBU (8.4microliters (μL), 0.056 mmol) was introduced and the solution wasstirred at room temperature for about 3.5 hours, followed by addition ofbenzoic acid (e.g., at 30.0 mg) for quenching. The examplemannose-C₂GuanBoc was then purified by precipitation twice in hexane atroom temperature.

Initiations of OROP via other acetonide protected monosaccharides,including diacetone-D-galactose, diacetone-D-glucose, anddiacetone-D-fructosc with MTC-C₂GuanBoc were conducted similarly to theabove protocol to generate galactose-C₂GuanBoc, glucose-C₂GuanBoc, andfructose-C₂GuanBoc, and the resulting polymers were purified byprecipitation in hexane.

The example mannose-C₂GuanBoc polymer prepared in accordance withsynthesis scheme 401 and the above noted protocol demonstrated a yieldof 0.41 grams (g) or (81%). The results of a proton nuclear magneticresonance (¹H NMR) study at 400 megahertz (MHz), in deuteratedchloroform (CDCl₃) at 22 degrees Celsius (° C.), provided the following:11.70-11.33 (m, 18H, [—NHCOOC(CH₃)₃]), 9.18-8.40 (m, 18H, —CH₂NHC—),6.05-4.74 (m, 3H, protons from diacetone-D-mannose), 4.46-4.10 (m, 110H,—OC₂H₄OCH—, —COOCH₂CH₂— and proton from diacetone-D-mannose), 3.88-3.62(m, 36H, —COOCH₂CH₂—), 1.75-1.65 (m, 12H, [—C(CH₃)₂]₂, 1.55-1.41 (m,342H, [—COOC(CH₃)₃]₂), 1.29-1.05 (m, 57H, —CH₃). A disparity (Ð) ofÐ=1.17 was observed.

The example galactose-C₂GuanBoc polymer prepared in accordance withsynthesis scheme 401 and the above noted protocol has the followingchemical properties: ¹H NMR (400 MHz, CDCl₃, 22° C.): 12.29-10.68 (m,19H [—NHCOOC(CH₃)₃]), 8.96-8.33 (m, 19H, —CH₂NHC—), 6.02-4.73 (m, 3H,protons from Diacetone-D-galactose), 4.50-3.91 (m, 118H, —OC₂H₄OCH—,—COOCH₂CH₂— and protons from Diacetone-D-galactose), 3.76-3.65 (m, 38H,—COOCH₂CH₂—), 1.76-1.60 (m, 12H, [—C(CH₃)₂]₂, 1.55-1.39 (m, 342H,[—COOC(CH₃)₃]₂), 1.29-1.17 (m, 54H, —CH₃), Ð=1.16.

The example glucose-C₂GuanBoc polymer prepared in accordance withsynthesis scheme 401 and the above noted protocol has the followingchemical properties: ¹H NMR (400 MHz, CDCl₃, 22° C.): 11.70-11.33 (m,16H [—NHCOOC(CH₃)₃]), 9.18-8.40 (m, 16H, —CH₂NHC—), 6.03-4.77 (m, 3H,protons from Diacetone-D-glucose), 4.46-4.10 (m, 100H, —OC₂H₄OCH—,—COOCH₂CH₂— and proton from Diacetone-D-glucose), 3.88-3.62 (m, 32H,—COOCH₂CH₂—), 1.75-1.65 (m, 12H, [—C(CH₃)₂]2, 1.55-1.41 (m, 288H,[—COOC(CH₃)₃]₂), 1.29-1.05 (m, 48H, —CH₃). Ð=1.20.

The example fructose-C₂GuanBoc polymer prepared in accordance withsynthesis scheme 401 and the above noted protocol has the followingchemical properties: ¹H NMR (400 MHz. CDCl₃, 22° C.): 11.10-10.82 (m,17H [—NHCOOC(CH₃)₃]), 8.66-8.30 (m, 17H, —CH₂NHC—), 6.13-4.68 (m, 3H,protons from diacetone-D-fructose), 4.55-3.86 (m, 106H, —OC₂H₄OCH—,—COOCH₂CH₂— and protons from diacetone-D-fructose), 3.73-3.68 (m, 34H,—COOCH₂CH₂—), 1.70-1.61 (m, 12H, [—C(CH₃)₂]₂, 1.53-1.34 (m, 306H,[—COOC(CH₃)₃]₂), 1.30-1.16 (m, 51H, —CH₃). Ð=1.16.

Synthesis scheme 401 was further continued for mannose-C₂GuanBoc,galactose-C₂GuanBoc, glucose-C₂GuanBoc and fructose-C₂GuanBoc, whereinboth the acetonide and Boc protecting groups were removed in a one-stepreaction under acidic conditions in accordance with deprotection 416. Inthis regard, separately, the respective protected polymers were chargedin a glass vial with 1.0 mLof CH₂Cl₂ equipped with a stir bar, with theaddition of TFA (e.g., 2.0 ml) and H₂O (0.2 ml) under constant stirringat room temperature for 18 hours. The solvent of the reaction mixturewas subsequently removed in vacuo, and the residue was dialyzed againstacetonitrile/isopropanol (1:1 volume of solute to volume of solution(v/v)) in a dialysis bag with molecular weight cut-off (MWCO) of 1000 Da(Spectra/Por 7, Spectrum Laboratories Inc.) for 24 hours. Finally, thesolvent within the dialysis bag was removed under vacuum.

Because the boc protecting groups can easily be removed under TFAconditions, the deprotection reactions were highly efficient where fulldeprotection was observed. The catalytic addition of a drop of water tothe reaction environment allowed for mild and effective removal of theacetonide protecting group from the sugar initiator. (e.g., asdetermined by ¹H NMR spectroscopy). To enhance clarity, the resultantdeprotected polymers having Structure 418 and prepared in accordancewith synthesis scheme 401 and the protocol described above, are referredto herein according to their respective sugar initiators, as well as thedegree of polymerization after deprotection and dialysis. In thisregard, the resultant antimicrobial polymers (having Structure 418 orthe like) are respectively referred to herein as mannose_16,galactose_18, glucose_17, and fructose_16. For example, mannose_16denotes a homopolymer containing 16 repeating units of deprotectedguanidine side groups with a mannose furanose initiator, whilefructose_16 denotes a polymer of similar chain length containing afructose initiator, and so on.

The resultant mannose_16 obtained was a white sticky polymer at about an80% yield or more. The ¹H NMR performed at 400 MHz, in dimethylsulfoxide (DMSO_(d),) at 22° C. demonstrated the following chemicalproperties: 7.73-7.00 (m, 64H, —CH₂NHC═NHNH₂), 5.20-5.04 (m, 2H,anomeric protons from mannose furanose), 4.79-4.16 (m, 68H, —OC₂H₄OCH—and 4 protons from mannose furanose), 4.15-4.03 (m, 32H, —COOCH₂CH₂—),1.37-1.06 (m, 48H, —CH₃). A yield of greater than 85% was obtained forexample galactose_18. The ¹H NMR for galactose_18 (also performed at 400MHz DMSO_(d) at 22° C.) demonstrated the following chemical properties:7.75-6.68 (m, 72H, —CH₂NHC═NHNH₂), 5.12-5.05 (m, 2H, anomeric protonsfrom galactose), 4.64-4.16 (m, 76H, —OC₂H₄OCH— and 4 protons fromgalactose), 4.15-4.03 (m, 36H, —COOCH₂CH₂—), 3.56-3.52 (m, 2H, H_(β) ofgalactose), 3.50-3.39 (m, 36H, —COOCH₂CH₂—), 1.26-1.08 (m, 54H, —CH₃).The example, antimicrobial polymer glucose_17 has the followingproperties: (>80% yield). ¹H NMR (400 MHz, DMSO_(d), 22° C.): 7.67-6.89(m, 64H, —CH₂NHC═NHNH₂), 5.15-5.07 (m, 2H, anomeric protons fromglucose), 4.48-4.17 (m, 72H, —OC₂H₄OCH— and 4 protons from glucose),4.16-4.00 (m, 34H, —COOCH₂CH₂—), 1.29-1.18 (m, 51H, —CH₃). The examplefructose_16 has the following properties: (>85% yield). ¹H NMR (400 MHz,DMSO_(d), 22° C.): 7.69-6.85 (m, 64H, —CH₂NHC═NHNH₂), 5.15-5.08 (m, 2H,anomeric protons from galactose), 4.97-4.16 (m, 68H, —OC₂H₄OCH— and 4protons from galactose), 4.14-4.02 (m, 32H, —COOCH₂CH₂—), 3.59-3.51 (m,2H, H_(β) of fructose), 3.49-3.38 (m, 32H, —COOCH₂CH₂—), 1.29-1.06 (m,48H, —CH₃).

The guanidinium-based macromolecules disclosed herein, includingguanidinium-based macromolecules having Structure 100, Structure I,Structure II, Structure III, Structure IV, Structure 418, mannose_16,galactose_18, glucose 17, fructose_16, the like, have demonstratedstrong efficacy as antimicrobial agents against both Gram-negative andGram-positive microbes, including MDR microbes, such as but not limitedto: K pneumoniae. A. baumannii, Escherichia coli (E. coli),Staphylococcus aureus (S. aureus), methicillin-resistant S. aureus(MRSA), and Pseudomonas aeruginosa (P. aeruginosa). The subjectguanidinium-based macromolecules have also demonstrated high efficacy(e.g., a 100% bacterial growth inhibition rate) at relatively loweffective dose (ED) amounts (e.g., an ED₅₀ less than 7.0milligrams/kilogram (mg/kg) and an ED₉₅ less than 21.0 mg/kg), no (orextremely low) toxicity (e.g., as demonstrated via a RBC viability levelgreater than 95% at an effective dose), and strong immunity todevelopment of antimicrobial resistance.

In this regard, the efficacy, toxicity and antimicrobial resistancedevelopment characteristics of the subject guanidinium-basedmacromolecules (e.g., particularly mannose_16 and glucose_17) was testedin vitro and in vivo (e.g., in a mouse model), against clinicallyisolated bacterial strains, including MDR K. pneumoniae, E. coli, A.baumannii, and MRSA strains. To facilitate comparison, an antimicrobialguanidinium-based macromolecule without a sugar functional group(identified herein as Dp20), as well as strong antibiotics (includingimipenem, vancomycin, ceftriaxone, and gentamycin), were also testedagainst the clinically isolated pathogens. The MDR clinical isolateswere obtained from patients' blood and phlegm. All isolates wereidentified by routine laboratory methods and stored in 20% (v/v)glycerol at 80□. The in vitro tests are described with reference toFIGS. 5-8, and the in vivo tests are described with reference to FIGS.9-11.

FIGS. 5 and 6 respectively illustrate the MICs of mannose_16 andglucose_17 relative to Dp20 and antibiotics (ceftriaxone, gentamycin,imipenem, and vancomycin) against the clinical isolates mentioned above.The MICs of mannose_16, glucose_17, Dp20 and the respective antibioticsagainst the clinical isolates were measured via the broth microdilutionmethod. In accordance with the broth microdilution method, MDR microbesincluding one or more strains of K. pneumoniae. E. coli. A. baumannii,and MRSA were harvested in midexponential growth phase after grownovernight in Mueller-Hinton (MH) agar plates at 37′. The antimicrobialagents were prepared in Mueller Hinton broth (MHB) at variousconcentrations. The bacteria suspensions were then diluted withphosphate-buffer saline (PBS), with a potential of Hydrogen (PH) of 7.4)to adjust the turbidity approximately to the Standard McFarland 0.6,which corresponds to the concentration of 1×10⁸ colony-forming unit(CFU) per mL (CFU/mL, after which the bacteria suspension was furtherdiluted by 100-fold with MHB (1×10⁶ CFU/mL). Subsequently, equal volumes(100 μL) of bacterial suspension and agent solution prepared previouslywere mixed in each well of a 96-well plate and incubated for 18 hours at37° C. Broth containing bacteria alone was employed as the negativecontrol, and each MIC was tested in triplicate. The results arepresented in FIGS. 5 and 6.

In this regard. FIG. 5 presents an example, non-limiting table 500providing the cumulative distribution of MIC values (in μg/mL) for Dp20,mannose_16, glucose_17, ceftriaxone, gentamycin, and imipenem against 10clinically isolated MDR A. baumannii strains. As reported in FIG. 5, theMIC values represent the lowest concentration of the testedantimicrobial agents at which no visible turbidity was observed withunaided eyes, or 100% bacterial growth inhibition. The MIC valuesmeasured range from 1.0 (μg/mL) to 512 μg/mL or greater (exponentiallyincreased) and are presented at the top of chart 500 along the x-axis.The values provided within the respective cells of table 500 andcorrespond to different percentages of bacterial strains observed at therespective MIC values. In this regard, a value of 100 for Dp20corresponds to 100% bacterial strains (e.g., 10 out of 10 strains) whichhave the particular MIC value (64 μg/mL) or below, a value of 90corresponds to 90% bacterial strains (e.g., 9 out of 10 strains) whichhave the particular MIC value (32 μg/mL) or below, a value of 60corresponds to 60% bacterial strains (e.g., 6 out of 10 strains) whichhave the particular MIC value (16 μg/mL), and so on.

As shown in table 500, to inhibit the growth of 10 MDR A. baumanniistrains, 64.0, 16.0 and 32.0 μg/mL is needed for DP20, mannose_16 andglucose_17, respectively. Accordingly, antimicrobial guanidinium-basedpolymers initiated from either mannose or glucose have shownsubstantially higher activity than guanidinium polymers without thecarbohydrate targeting moiety and the antibiotic controls.

FIG. 6 presents an example, non-limiting table 600 comparing the MICvalues (in micrograms (μg)/mL) of the tested antimicrobial agentsagainst different types and strains of clinically isolated MDR bacteria.As reported in FIG. 6, the MIC values represent the lowest concentrationof the tested antimicrobial agents at which no visible turbidity wasobserved with unaided eyes, or 100% bacterial growth inhibition. Asshown in table 600, mannose_16 and glucose_17 demonstrated high efficacyagainst each of the tested bacterial strains with MIC values rangingfrom only 4.0 μg/mL to 32.0 μg/mL. In this regard, both mannosc_16 andglucose_17 demonstrated broad-spectrum efficacy against Gram-negativeand Gram-positive strains, whereas imipenem only demonstrated efficacyagainst the Gram-negative strains (e.g., the K. pneumoniae (KP), E. coli(EC), and A. baumannii (AB) strains), and vancomycin only demonstratedefficacy Gram-positive strains (e.g., the methicillin-resistant S.aureus (MSRA) strains). In addition, the MIC values for mannose_16 andglucose_17 are substantially lower than those of Dp20 with respect toeach of the different bacterial strains. Further, the MIC values formannose_16 and glucose_17 are substantially lower than the MIC valuesfor imipenem with respect to strains KP7958, KP9170, AB4123 and AB10361.

FIG. 7 illustrates an example, non-limiting graph 700 that depictshemolysis activity of one or more guanidinium macromolecules inaccordance with one or more embodiments described herein. In thisregard, the RBC hemolytic activity of guanidinium-based macromoleculeshaving structure 100 with various sugar moieties (lactate, glucose,mannose and galactose) as the targeting moiety R were tested at variousconcentrations (measured in μg/mL). As shown in FIG. 7, at polymerconcentrations within the therapeutic dose (e.g., 64 μg/mL or less), nohemolytic activity (e.g., as shown via a RBC viability level of 100%)was demonstrated by the glucose functionalized guanidinium basedpolymers and extremely low hemolytic activity was demonstrated by thepolymers including the other sugar moieties (e.g., as shown via the RBCviability level at about 95% or greater). This low hemolytic activitywas further demonstrated for each of the different sugar moiety basedpolymers far past the therapeutic dose. For example, even atconcentrations up to 1000 μg/mL, the respective polymers demonstratedRBC viability greater than 80%. Accordingly, the disclosed guanidiniummacromolecules with different sugar targeting moieties are thus highlyselective towards bacterial cells (e.g., as opposed to RBCs) andnontoxic even at the high polymer concentrations.

FIG. 8 illustrates an example, non-limiting graph 800 that demonstratesthe robustness of one or more guanidinium macromolecules againstbacteria resistance development in accordance with one or moreembodiments described herein. In this regard, A. baumannii 4123 wasemployed to study the propensity to develop resistance againstmannose_16 and glucose_17, as well as Dp20, imipenem and levofloxacinfor comparison. To test bacterial resistance and resistance prevention,a sub-lethal dose (so as to not kill all bacteria) of the respectiveantimicrobials was repeatedly applied to A. baumannii samples for 24hours at each passage, wherein the bacteria were allowed to replicateand/or mutate. The MIC values of the respective antimicrobials were thenmeasured after each repetition to monitor MIC changes. An increase inMIC value indicates development of resistance in the bacteria. Inparticular, samples of A. baumannii bacteria were first exposed to therespective antimicrobials (e.g., mannose_16 and glucose_17, Dp20,imipenem and levofloxacin) for the determination of MICs as describedabove. Subsequently, 50.0 μL of bacteria from wells of 0.5×MIC wereharvested and grown overnight, and then subjected to MIC determinationsfor up to 20 similar serial passages (e.g., single applications once aday for 20 days).

The development of resistance to the antimicrobial agents in A.baumannii was monitored by recording the changes in the MIC as shown viagraph 800. The three horizontal lines at the bottom of graph 800respectively correspond to Dp20, glucose_17 and mannose_16. As shown viagraph 800, repeated use of the sugar-functionalized polymers glucose_17and mannose_16, (as well as Dp20), did not cause resistance in A.baumannii 4123, while the bacteria with multiple treatments withimipenem or levofloxacin developed significant resistance. Therespective lines at the bottom of graph 800 for Dp20, glucose_17 andmannose_16 are shown slightly above the zero MIC value for illustrativepurposes. However, in practice each of the measured MIC values for Dp20,glucose_17 and mannose_16 remained constant (i.e. MICn/MIC₀=1, whereMICn represents MIC at n passage, and MIC₀ represents MIC at 0 passage).

In addition to the in vitro studies discussed above, FIGS. 9-11demonstrate the antimicrobial efficacy of the subject guanidinium-basedmacromolecules (e.g., particularly mannose_16 and glucose_17) as testedrelative to Dp20 and imipenem in a mouse model (e.g., in vivo). In thisregard, immunosuppressed mice were used for the following in vivostudies. Immunosuppression was induced by intraperitoneal injection of200 mg/kg cyclophosphamide 4 days prior to infection. The mice wereanesthetized by intraperitoneal injection of 1% pentobarbital (40 mg/kg,sigma). The mice were then infected with A. baumannii 4123 to determinethe minimum lethal dose for subsequent effective dose and efficacytesting. Briefly, overnight cultures of A. baumannii 4123 were harvestedand suspended in phosphate-buffered saline (PBS). Subsequently, thecyclophosphamide-pretreated mice were injected with 0.3 mL of thebacterial suspension at designated doses (e.g., 1.0×10⁸, 2.5×10⁸, 5×10⁸,1.0×10⁹, 2.0×10⁹ CFU/mL, six mice per group) via the tail vein. Theminimum lethal dose, the lowest concentration sufficient to cause 100%mortality, was determined from the survival rate of mice at 48 hourspost-infection by the BLISS method.

The effective doses ED₅₀, (the effective dose that cures 50% infectedmice), and ED₉₅. (the effective dose that cures 95% infected mice), ofmannose_16 and glucose_17, and Dp20 and imipenem for comparison, weretested using an A. baumannii 4123-caused bacteremia infection mousemodel as follows. Firstly, a bacterial suspension with the minimumlethal dose (0.3 mL) was introduced to the above describedcyclophosphamide-pretreated mice via the tail vein. The antimicrobialagents (e.g., mannose_16, glucose_17, Dp20 and imipenem) were thenrespectively administered to different groups of mice (six mice pergroup) intraperitoneally, 1.0 and 6.0 hours after infection atdesignated doses (e.g., 1.0, 2.5, 5.0, 7.5, 10.0, 15.0, 20.0 mg/kg formannose_16, glucose_17. Dp20 and imipenem, 0.2 mL/20 g). The number ofsurviving mice in each group was recorded for 7.0 days to estimate ED₅₀and ED₉₅ via the BLISS method. The results are provided in FIG. 9.

In particular, FIG. 9 presents an example, non-limiting table 900demonstrating the results of the above described effective doseevaluation of various antimicrobials, including the subjectguanidinium-based macromolecules, against A. baumannii 4123 in the bloodinfected mouse model, in accordance with one or more embodimentsdescribed herein. As shown in table 900, the mannose functionalpolycarbonate (e.g., mannose_16) and the glucose functionalpolycarbonate (glucose_17) respectively demonstrate low ED₅₀ and ED₉₅values (e.g., less than 7.0 mg/kg for ED₅₀ and less than 21 for ED₉₅mg/kg). In this regard, both mannose_16 and glucose_17 are highlyeffective against A. baumannii in vivo at relatively low effectivedoses. In addition, mannose_16 exhibits a lower ED₅₀ and ED₉₅ thanimipenem and Dp20, demonstrating improved in vivo efficacy. Furthermoreglucose_17 exhibits a lower ED₅₀ than Dp20 and a lower ED₉₅ thanimipenem and Dp20.

In addition to the ED₅₀ and ED₉₅ evaluations described above, theefficacy of mannose_16 and glucose_17 was also tested for effectivenessover time using the immunosuppressed mice described above. In thisregard, immunosuppressed mice were randomly divided into five groups, 25in each group. The immunosuppressed mice were then injected with 1.5×10⁸CFUs of A. baumannii 4123 via the tail vein. Then each group of micewere injected intraperitoneally with either imipenem, dp20, mannose_16,glucose_17 or PBS, 1.0 hour and 6.0 hours after infection at doses ofED₅₀ (e.g., 6.1 mg/kg for imipenem, 8.3 mg/kg for dp20, 5.6 mg/kg formannose_16 and 6.6 mg/kg for glucose_17). The PBS-treated group was usedas the control. At 1.0 hour, 4.0 hours, 8.0 hours and 24 hourspost-infection, five anesthetized mice in each group were sacrificed andblood samples were obtained from the periorbital plexus. The bloodsamples were subsequently serially diluted and plated on an MH agarplate. After being incubated at 37° C. overnight, the number of viablebacteria was counted. The results are presented in FIG. 10. Further, at24 hours post infection, 5.0 mice in each group were sacrificed toacquire their organs including liver, spleen, kidney and lung. Obtainedorgans were homogenized in 2.0 mL of PBS. The homogenate samples werealso serially diluted and plated on MH agar plates and incubatedovernight at 37° C. The number of bacterial colonies in the respectivesamples was then counted. The results were showed as mean Log 10 (Lg)(CFU/mL)±standard deviation (SD), and are presented in FIG. 11.

FIG. 10 presents an example, non-limiting graph 1000 demonstrating theresults of the above described mouse model testing the efficacy ofvarious antimicrobials, including the subject guanidinium-basedmacromolecules, against A. baumannii over time. In this regard, graph1000 charts the bacterial count observed in the infected mice as afunction of time (e.g., tested at the 1.0, 4.0, 8.0, and 24-hour marks)following the administration two doses (one at 1.0 hour post infectionand another at 6.0 hours post infection) of the therapeutics imipenem.Dp20, mannose_16 and glucose_17. As shown in graph 1000, the glucose andmannose-based polymers led to a rapid and significant decrease inbacterial count relative to the optimized antibiotic imipenem.Mannose_16 and glucose_17 also demonstrated a more rapid and overallhigher decrease in bacterial count over time relative to Dp20.

FIG. 11 presents an example, non-limiting graph 1000 demonstrating theresults of the above described mouse model testing the efficacy ofvarious antimicrobials, including the subject guanidinium-basedmacromolecules, against A. baumannii over time. In this regard. graph1100 particularly compares the bacterial count remaining in variousorgans of A. baumannii infected mice after a period of 24 hours and twodoses (one at 1.0 hour post infection and another at 6.0 hours postinfection) of either imipenem. Dp20, mannose_16, or glucose_17. As shownin graph 1100, mannose_16 proved most effective and glucose_17 provedsecond most effective at killing the bacteria relative to the othertested antimicrobials, regardless of the organ of infection. In thisregard, clearly the sugar-decorated therapeutics manifestedsignificantly lower bacteria counts in the liver, kidney and spleenrelative to imipenem.

The various structures (e.g., described regarding FIGS. 1-3, 4A and 4B),compositions (e.g., described regarding FIGS. 1-11), and/or methods(e.g., described regarding FIGS. 4A, 4B and FIGS. 5-11) described hereincan regard chemical compounds that can be incorporated into a variety ofapplications. For example, said applications can include cleaning,sanitizing, disinfecting, and/or otherwise treating various articlessuch as, but not limited to: food packaging, medical devices, floorsurfaces, furniture surfaces, wound care instruments (e.g., bandagesand/or gauss), building surfaces, plants (e.g., agricultural crops),ground surfaces, farming equipment, beds, sheets, clothes, blankets,shoes, doors, door frames, walls, ceilings, mattresses, light fixtures,facets, switches, sinks, grab rails, remote controls, vanities, computerequipment, carts, trolleys, hampers, bins, a combination thereof, and/orthe like. In another example, said applications can includepharmaceuticals, pharmaceutical salts, hygiene products (e.g., soapsand/or shampoos), and/or the like. In a further example, saidapplications can include agricultural sprays and/or aqueous solutionsthat can facilitate processing crops for consumption.

FIGS. 12-14 illustrate various methodologies in accordance with thedisclosed subject matter. While, for purposes of simplicity ofexplanation, the methodologies are shown and described as a series ofacts, it is to be understood and appreciated that the disclosed subjectmatter is not limited by the order of acts, as some acts can occur indifferent orders and/or concurrently with other acts from that shown anddescribed herein. For example, those skilled in the art will understandand appreciate that a methodology could alternatively be represented asa series of interrelated states or events, such as in a state diagram.Moreover, not all illustrated acts can be required to implement amethodology in accordance with the disclosed subject matter. Repetitivedescription of like elements employed in respective embodiments isomitted for sake of brevity.

FIG. 12 illustrates a flow diagram of an example, non-limiting method1200 that can facilitate generating one or more antimicrobialguanidinium macromolecules in accordance with one or more embodimentsdescribed herein. At 1202, a monosaccharide can be dissolved with aguanidinium functionalized monomer in a solvent. As a result, at 1204the guanidinium functionalized monomer can be polymerized to form apolymer, wherein the polymer comprises a plurality of covalently bondedunits of the guanidinium functionalized monomer and a least twoperipheral ends, wherein at least one unit of the monosaccharide iscovalently bound to at least one of the peripheral ends.

FIG. 13 illustrates a flow diagram of another example, non-limitingmethod 1300 that can facilitate generating one or more guanidiniummacromolecules in accordance with one or more embodiments describedherein. At 1302, a protected monosaccharide can be dissolved with aguanidinium functionalized monomer in a solvent, wherein the guanidiniumfunctionalized monomer comprises cyclic carbonate with a protectedguanidinium moiety bound to the cyclic carbonate via a spacer group. At1304, an organocatalyzed ring opening polymerization of the cycliccarbonate can be initiated using the protected monosaccharide to form afirst polymer, wherein the first polymer comprises a plurality ofcovalently bonded units of the guanidinium functionalized monomer and aleast two peripheral ends, wherein at least one unit of the protectedmonosaccharide is covalently bound to at least one of the peripheralends. At 1306, protection groups from the protected monosaccharide andthe protected guanidinium moiety can be removed, thereby forming asecond polymer, wherein the second polymer is effective at killingGram-negative bacteria and Gram-positive bacteria.

FIG. 14 illustrates a flow diagram of an example, non-limiting method1400 that can facilitate killing of a pathogen with one or moreguanidinium macromolecules in accordance with one or more embodimentsdescribed herein. At 1402, a MDR (MDR) microbe with a chemical compound(e.g., an antimicrobial guanidinium-based macromolecule having Structure100, Structure, I, Structure II, Structure III, Structure IV,mannose_16, glucose_17, galactose_18, fructose_16, and the like). Inthis regard, the chemical compound comprises a polymer backbone, one ormore guanidinium moieties that extend from the polymer backbone, and acarbohydrate moiety that extends from a peripheral end of the polymerbackbone. At 1404, the method further comprises, disrupting a membraneof the MDR microbe in response to the contacting. For example, in one ormore implementations, the carbohydrate moiety can selectively attractthe microbe, resulting in consumption of the macromolecule by themicrobe. The macromolecule is then released through the membrane leadingto cytosol precipitation and subsequent bacterial cell apoptosis occurs.Moreover, the carbohydrate targeting moieties that comprise sugars havebeen shown to target the bacteria cell wall/membrane in such a way thatthe sugar gets taken up and chemically incorporated into the bacterialcell wall/membrane. Accordingly, in addition to facilitating targetingselectivity and antimicrobial efficiency of the guanidinium basedmacromolecule, the sugar moiety can further contribute to local disorderand stress within the lipid bilayer of the bacterial cell, leading tothe membrane damage and thus a higher rate of cell lysis/apoptosis.

In addition, the term “or” is intended to mean an inclusive “or” ratherthan an exclusive “or.” That is, unless specified otherwise, or clearfrom context. “X employs A or B” is intended to mean any of the naturalinclusive permutations. That is, if X employs A; X employs B; or Xemploys both A and B, then “X employs A or B” is satisfied under any ofthe foregoing instances. Moreover, articles “a” and “an” as used in thesubject specification and annexed drawings should generally be construedto mean “one or more” unless specified otherwise or clear from contextto be directed to a singular form. As used herein, the terms “example”and/or “exemplary” are utilized to mean serving as an example, instance,or illustration. For the avoidance of doubt, the subject matterdisclosed herein is not limited by such examples. In addition, anyaspect or design described herein as an “example” and/or “exemplary” isnot necessarily to be construed as preferred or advantageous over otheraspects or designs, nor is it meant to preclude equivalent exemplarystructures and techniques known to those of ordinary skill in the art.

What has been described above include mere examples of systems,compositions, and methods. It is, of course, not possible to describeevery conceivable combination of reagents, products, solvents, and/orarticles for purposes of describing this disclosure, but one of ordinaryskill in the art can recognize that many further combinations andpermutations of this disclosure are possible. Furthermore, to the extentthat the terms “includes,” “has,” “possesses,” and the like are used inthe detailed description, claims, appendices and drawings such terms areintended to be inclusive in a manner similar to the term “comprising” as“comprising” is interpreted when employed as a transitional word in aclaim. The descriptions of the various embodiments have been presentedfor purposes of illustration, but are not intended to be exhaustive orlimited to the embodiments disclosed. Many modifications and variationswill be apparent to those of ordinary skill in the art without departingfrom the scope and spirit of the described embodiments. The terminologyused herein was chosen to best explain the principles of theembodiments, the practical application or technical improvement overtechnologies found in the marketplace, or to enable others of ordinaryskill in the art to understand the embodiments disclosed herein.

What is claimed is:
 1. An antimicrobial macromolecule, comprising: apolymer backbone; one or more guanidinium moieties that extend from thepolymer backbone; and a targeting moiety that extends from the polymerbackbone.
 2. The antimicrobial macromolecule of claim 1, wherein thetargeting moiety comprises a substance favored for consumption bybacteria.
 3. The antimicrobial macromolecule of claim 1, wherein thetargeting moiety comprises a monosaccharide.
 4. The antimicrobialmacromolecule of claim 1, wherein the targeting moiety is selected froma group consisting of mannose and glucose.
 5. The antimicrobialmacromolecule of claim 1, wherein the polymer backbone comprisespolycarbonate.
 6. The antimicrobial macromolecule of claim 1, whereinthe polymer backbone is selected from a group consisting of polylysine,polyionene, and polyethylene imine.
 7. The antimicrobial macromoleculeof claim 1, wherein the antimicrobial macromolecule has a structurecharacterized by Structure I:

wherein R represents the targeting moiety, and wherein n represents aninteger greater than or equal to one and less than or equal to onethousand.
 8. The antimicrobial macromolecule of claim 1, wherein theantimicrobial macromolecule is effective at killing Gram-negativebacteria and Gram-positive bacteria.
 9. The antimicrobial macromoleculeof claim 1, wherein the antimicrobial macromolecule is effective atkilling MDR bacteria selected from a group consisting of: Acinetobacterbaumannii, Klebsiella pneumonia, Escherichia coli, Staphylococcusaureus, methicillin-resistant Staphylococcus aureus, and Pseudomonasaeruginosa.
 10. The antimicrobial macromolecule of claim 1, wherein theantimicrobial macromolecule exhibits low toxicity in mammalian cellsdemonstrated by a red blood cell viability level greater than 95% at aneffective dose.
 11. The antimicrobial macromolecule of claim 1, whereinthe antimicrobial macromolecule is attributed to no development ofbacterial antimicrobial resistance over at least 20 passages.
 12. Theantimicrobial macromolecule of claim 1, wherein the antimicrobialmacromolecule is biocompatible.
 13. The antimicrobial macromolecule ofclaim 1, wherein the antimicrobial macromolecule is biodegradable. 14.An antimicrobial polymer comprising a structure characterized byStructure I:

wherein R comprises a carbohydrate moiety, wherein n comprises aninteger greater than or equal to one and less than or equal to onethousand, and wherein X comprises a spacer group selected from a groupconsisting of: a propyl group, a butyl group, a pentyl group, acyclohexyl group, a phenyl group, and a benzyl group.
 15. Theantimicrobial polymer of claim 14, wherein the carbohydrate moiety isselected from a group consisting of mannose, glucose, fructose andgalactose.
 16. The antimicrobial polymer of claim 14, wherein theantimicrobial polymer is effective at killing Gram-negative bacteria andGram-positive bacteria.
 17. A method, comprising: dissolving amonosaccharide with a guanidinium functionalized monomer in a solvent;and polymerizing the guanidinium functionalized monomer to form apolymer, wherein the polymer comprises a plurality of covalently bondedunits of the guanidinium functionalized monomer and at least twoperipheral ends, wherein at least one unit of the monosaccharide iscovalently bound to at least one of the peripheral ends.
 18. The methodof claim 17, wherein the monosaccharide comprises a protectedmonosaccharide and wherein the guanidinium functionalized monomercomprises cyclic carbonate with a protected guanidinium moiety bound tothe cyclic carbonate via a spacer group.
 19. The method of claim 18,wherein the polymerizing comprises an organocatalyzed ring openingpolymerization of the cyclic carbonate using the protectedmonosaccharide as an initiator.
 20. The method of claim 18, wherein themethod further comprises: removing protection groups from the protectedmonosaccharide and the protected guanidinium moiety, thereby forming adeprotected polymer, wherein the deprotected polymer is effective atkilling Gram-negative bacteria and Gram-positive bacteria.